The overall objective of the proposed study is to elucidate the mechanism of specificity and regulation of gamma-secretase and provide a molecular basis for developing specific inhibitors to treat Alzheimer's disease (AD) and other human disorders. Gamma-secretase cleaves amyloid precursor protein (APR) to generate the C-termini of A-beta peptides (A-beta40 and A-beta42), the final step of amyloid production. A-beta, the major constituent of amyloid plaques found in AD, is believed to play a critical role in the neuropathogenesis of AD. It is known that A-beta42 is more prone to aggregation than A-beta40, therefore biological or environmental factors that promote increased A-beta42 production may accelerate the pathological cascade leading to AD. Despite intensive studies on A-beta peptides and gamma-secretase, the molecular mechanism controlling the specificity of gamma-secretase activity for A-beta40 and Abeta42 production is unresolved. Proposed studies will offer a better understanding of the molecular basis for gamma-secretase specificity, in addition to providing critical insight into the pathogenesis of AD. The first aim of the proposed study is to identify novel proteins, in addition to presenilin, that contribute to the active site of gamma-secretase using active-site directed inhibitors. A second specific aim is to investigate the effect of naturally occurring familial AD mutants (APP and presenilin) on gamma-secretase activity to elucidate the molecular pathogenesis of AD. The third aim is to analyze different sizes of gamma-secretase complexes that possess varied activity for A-beta40 and A-beta42 production to define the structural and molecular basis of gamma-secretase specificity. These proposed studies will lead to a comprehensive understanding of gamma-secretase specificity. Moreover, these investigations will facilitate the development of potent and specific inhibitors of gamma-secretase for potential AD therapies.